1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)

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    Screening of Bitter gourd (Momordica charantia L.) genotypes for resistance to powdery mildew disease
    (Department of Vegetable Science, College of Agriculture, Vellanikkara, 2025-05-17) Chethan, S V; Prasanth, K
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    In-vitro chemotherapy for inducing tolerance towards Tomato leaf curl New Delhi virus (ToLCNDV)in bitter gourd (Momordica charanita L.)
    (Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, 2023-12-12) Adarsh, M V.; Smitha Bhasi
    The study entitled “In-vitro chemotherapy for inducing tolerance towards Tomato Leaf Curl New Delhi virus in bitter gourd (Momordica charantia L.)” was conducted at the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during 2022-23. The objective of the study was the evaluation of in-vitro treatment with antiviral compounds, viz., Ribavirin, Virus–Ex and extracts of Bougainvillea spectabilis for inducing tolerance towards Begomovirus- ToLCNDV in bitter gourd (Momordica charantia L.). The explants namely seed of bitter gourd variety Preethi was collected from, Instructional farm, College of agriculture, Vellayani. Seeds were subjected to hot water treatment at 55°C for 15 minutes, followed by washing in 0.2% bavistin for 15 minutes and kept in dark for inducing germination. 91.2% germination was noticed in 3 days. The germinated seeds were grown in Murashige and Skoog (MS) medium supplemented with 2ppm BA and 20ppm concentration of various antiviral compounds viz., Ribavirin (Treatment 1), Virus-Ex (Treatment 2), extract of Bougainvillea spectabilis (Treatment 3) along with control. Percentage of culture establishment was found to be maximum (65.25%) in ribavirin treated plants followed by virus ex and bougainvillea. 20 days old in-vitro grown plants were hardened in coir pith compost and the biometric observations were taken and compared. Significant increase in plant height and number of leaves over control was noticed in treatment using ribavirin and virus ex respectively. DNA was isolated from 20 day old (hardened) treated and control plants prior to whitefly transmission and were confirmed to be virus free using PCR. Whitefly mediated artificial inoculation of virus in hardened plants (25 day old) was done and the plants were maintained inside insect proof cage for symptom development. Symptoms were observed in control plants after 10 days of whitefly transmission. Further PCR was performed to confirm the presence/absence of Begomovirus. Absence of virus in treatments with ribavirin and virus ex were 59 confirmed in PCR. Treatment using extracts of bougainvillea did not show any symptoms but confirmed positive in molecular detection. The control plants which developed symptoms was found positive in PCR. Peroxidase assay (Lobenstein and Linsey method) showed maximum increase in activity (3-fold) in plants treated with ribavirin followed by extracts of bougainvillea (2-fold) three days after whitefly transmission. Biometric observations on 35th day viz., plant height, number of leaves and leaf area were found higher in all the treatments compared to control, out of which treatment using ribavirin was found to be highly significant for all the parameters. To conclude, the present study in bitter gourd could confirm the antiviral activity of ribavirin and virus ex towards Begomovirus through whitefly mediated transmission test and molecular detection using PCR
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    Entomopathogenic nematode for the management of cucumber moth, Diaphania indica (Saunders) (Lepidoptera: Crambidae) in bitter gourd
    (Department of Agricultural Entomology, College of Agriculture, Vellayani, 2023-04-12) Gayathri, P S; Nisha, M S
    The research work entitled “Entomopathogenic nematode for the management of cucumber moth, Diaphania indica (Saunders) (Lepidoptera: Crambidae) in bitter gourd” was carried out at Department of Entomology, College of Agriculture, Vellayani during 2020 to 2022. The objectives were to test the pathogenicity of entomopathogenic nematodes (EPNs) and to evaluate the effective EPN formulation against cucumber moth, D. indica in bitter gourd. The pathogenicity of two strains of entomopathogenic nematodes viz. Heterorhabditis indica Poinar and Steinernema carpocapsae Weiser were assessed against 3rd instar larvae of D. indica and Henosepilachna septima Dieke. Three different formulations viz. sponge, talc and alginate gel of the effective EPN strain were prepared and stored up to 11 weeks. Then, the survival percentage and virulence of infective juveniles (IJs) in each formulation was evaluated. A pot culture study was also conducted to evaluate the pathogenicity of effective EPN formulation against D. indica in bitter gourd. In the pathogenicity test, H. indica @ 100 IJs recorded highest mortality percentage of 42.50 for D. indica and 40.00 for H. septima at 24 Hours After Treatment (HAT). No mortality was observed for S. carpocapsae at all the concentration levels (10, 20, 50 and 100 IJs) for D. indica at 24 HAT. At 48 HAT, hundred per cent mortality was recorded only for H. indica at 100 IJs and it was on par with H. indica at 50 IJs. Both H. indica and S. carpocapsae at 50 and 100 IJs showed hundred per cent mortality at 60 HAT. All treatments showed hundred per cent mortality at 72 HAT. The number of IJs emerged from cadavers were slightly more for H. indica than S. carpocapsae for the same concentration of IJs. From the 1st experiment, H. indica was found to be more effective than S. carpocapsae against D. indica and H. septima. Therefore, survival percentage of IJs of H. indica were evaluated in different formulations. Highest survival percentage (100 per cent) was observed in alginate gel formulation in 1st week and it was on par with talc formulation up to 2 weeks. More than 50% survival of IJs was observed upto 8 weeks in alginate gel, 6 weeks in talc, 5 weeks in sponges and 3 weeks in water. 112 Results on virulence of IJs in different formulations revealed that IJs from alginate gel recorded mortality percentage ranging from 32.50 to 100.00 and 0.00 to 23.33 in 1st and 11th week of storage respectively against D. indica. It was statistically on par with freshly harvested juveniles at 72 HAT up to 5th week of storage. The next best formulation was found to be talc which was on par with freshly harvested juveniles at 72 HAT up to 4th week of storage. In the pot culture study, it was observed that the mortality of D. indica caused by IJs from alginate gel-based formulation of H. indica @ 4g L-1 (42.22 and 68.88) and talc- based formulation of H. indica @ 20g L-1 (40.00 and 68.88) was superior to talc-based formulation of Beauveria bassiana @ 2% (26.66 and 62.22) and neemazal 1% @ 0.2% + tween 80 (1%) (17.77 and 42.22) at 1 and 3 DAT respectively. The effect of H. indica was on par with talc-based formulation of B. bassiana @ 2% and neemazal 1% @ 0.2% + tween 80 (1%) at 5 and 7 DAT. At the same time, the highest mortality (100 per cent) was obtained for chlorantraniliprole 18.5SC@ 30g a.i. ha-1. The damage caused by D. indica ranged from 16.92 to 34.99 in alginate gel-based formulation of H. indica @ 4g L-1 and 19.97 to 41.74 in talc- based formulation of H. indica @ 20g L-1. Its effect on damage of leaves was on par with talc-based formulation of B. bassiana @ 2% of damage percentage 28.57, 40.21 and 41.00 at 3, 5, and 7 DAT respectively. Effect of H. indica formulations on the emergence of Bactrocera cucurbitae (Coquillett) from soil was studied under in vitro. Soil treated with alginate gel-based formulation of H. indica @ 4g L-1 and talc- based formulation of H. indica @ 20g L-1 recorded 16.66 per cent emergence of B. cucurbitae and its effect was on par with chlorantraniliprole 18.5SC@ 30g a.i. ha-1. Based on the present study, it can be concluded that H. indica can be stored in formulations viz. alginate gel and talc for better shelf life and it can be used as a biocontrol agent against D. indica, H. septima and B. cucurbitae in bitter gourd.
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    Elucidating the morpho - physiological and molecular changes during seed priming in bitter gourd (Momordica charantia L.)
    (Department of Seed Science and Technology, College of Agriculture, Vellanikkara, 2024-05-24) Thabsheer, P.; Rose Mary Francis
    A study to elucidate the morphological, biochemical and molecular changes leading to enhanced germination on priming in bitter gourd variety Preethi was conducted at College of Agriculture, Vellanikkara, Thrissur, during 2020-2023. Seeds of bitter gourd variety Preethi were dried to 6.84 per cent, and the quality parameters of the sourced seeds were assessed. They were divided into three equal sub-lots (5.0 kg each). Two sub-lots were packed in 700-gauge polyethylene bags and sealed airtight and one each stored for a period of six month under i). Ambient environment (Am.S), ii). Refrigerated storage (Rf.S) at ~18OC-20OC. The third sub-lot was packed in moisture-vapour proof aluminium foil bags seeds and stored for a period of six months at Medium term storage (MTS) facility at the ICAR-NBPGR (National Bureau of Plant Genetic Resources), Regional station, Vellanikkara. At the end of the storage period i.e., six months, the quality parameters of the stored seeds under the various storage environments were assessed following which they were further divided into 11 equal parts and subjected to 10 priming treatments (T1 to T10) following standard procedure, while unprimed seeds served as the control (T11). The priming treatments included T1: KNO3 0.3% for 2 h, T2: KNO3 150 ppm for 3 h, T3: KH2P04 10-2 M for 24 h, T4: PEG 6000 -1.5 MPa for 24 h, T5: GA3 100 ppm for 24 h, T6: Solid matrix priming with Perlite for 48 h, T7: Solid matrix priming with Cocopeat for 48 h, T8: Psuedomonas fluorescens 1x106 cfu.ml-1 for 24 h, T9: Hydration hot water at 50°C for 4 h and T10: Hydration cold water soaking for 24 h. Three separate studies as enumerated below were conducted to compare the effect of priming on germination of stored seeds under three different storage conditions viz. Ia) Effect of priming on seeds stored under ambient environment, Ib) Effect of priming on seeds stored in refrigerated storage (~18-20 OC) and Ic) Effect of priming on seeds stored in medium-term storage (~ 4-6 OC). Seed quality parameters viz., number of days to emergence, speed of germination, germination per cent, seedling root length (cm), seedling shoot length (cm), seedling dry weight (g), seed moisture content (%), germination capacity (GC), speed of germination, mean germination time (MGT), time taken for 50% germination (T50), energy of germination (GE), germination index (GI), coefficient velocity of germination (CVG) and seedling vigour indices were assessed after priming treatments in each experiment. In addition to the physiological observations, biochemical parameters viz., total soluble sugar (%), total protein (%), total oil content (%), H2O2 content (μmol g_1), lipid peroxidation (MDAμmol g_1), total dehydrogenase activity (OD), α–amylase activity (μmol g_1), catalase activity (μmol g_1), electrical conductivity of seed leachate (μScm-1), leakage of amino acid (μg leucine eqiv.ml -1) and leaching of sugar (μg glucose eqiv.ml -1) were recorded along with seed structural parameters viz., total seed coat thickness (μm), thickness of testa (μm), thickness of tegmen (μm), embryo length (μm), endosperm length (μm), endosperm breadth (μm), seed length (μm) and relative water absorption (RWA). Comparison of quality parameters before and after storage under the three environments pointed out that in addition to a reduction in germination, root length and shoot length the seedlings, the vigour indices I and II had also decreased in comparison to the estimates recorded prior to storage, while parameters like MGT and T50 of stored seeds were observed to have increased after six months of storage. The per cent reduction of speed of gemination, germination per cent, seedling root length, seedling shoot length, seedling dry weight, GC, GE, GI, CVG and vigour indices was however, the least in seeds stored in Am.S condition. Hence, it became evident that the per cent deterioration in seed stored at low temperatures is low, conversely, it reinforced the fact that storing bitter gourd seeds at low temperatures (MTs and Rf.S) would help maintain seed quality longer. The effect of seed priming treatments on germination (%) after six months of storage under i) S1: Ambient storage (Am.S), ii) S2: Refrigerated storage (Rf.S) and iii) S3: Medium-term storage (MTS) was anlaysed separately, each following a Completely randomized design with three replication and 11 priming treatments, in order to assess the best priming treatment under each storage environments studied. Germination of seeds stored under varying environmental conditions were found to be significantly influenced by the priming treatment they were subjected to. Germination of aged seeds from the three storage environments when subjected to Solid matrix priming with perlite (T6: Perlite for 48 h) cocopeat (T7: Cocopeat for 48 h) and hormonal priming with GA3 (T5: GA3 100 ppm for 24 h) were significantly superior over untreated seeds. Priming with PEG 6000 (T4: PEG 6000 -1.5 MPa for 24 h) was found to adversely affected germination irrespective of the storage environment the seeds were derived from. Although bio-priming seeds from Am.S with P. fluorescens (T8: P. f at 1x106 cfu.ml-1 for 24 h) and hydropriming (T9: Hydration hot water at 50°C for 4 h) was beneficial, it was not so in seeds derived from Rf.S and MTS. Similarly, hydration with hot water @ 50°C for 4 h was not found very beneficial in seeds stored under Rf.S and MTS as compared to Am.S. The impact of storage environment (S), priming treatment (T), and their interaction on physiological, biochemical and seed structural quality indices of seeds stored were analysed following a Completely randomized design with three replications and two factors viz., Factor I: Storage environments (S1, S2 and S3) and Factor II: Priming treatments (T1 to T11). Results indicated that, irrespective of the priming treatments, the quality indices of seed stored in MTS followed by Rf.S were found to be significantly superior to those stored under Am.S. It was evident that storing seeds at low temperatures (~ 18-20 0C in Rf.S, ~ 4-6 0C in MTS) was beneficial in bitter gourd. It resulted in higher germination as well as other physiological, biochemical and structural indices of the seed. Irrespective of the storage conditions, the quality indices of the aged seeds were found to be significantly influenced by the priming treatment they were subjected to. Solid matrix priming (SMP) with Perlite (T6: solid matrix priming with perlite for 48 h) invariably followed by solid matrix priming with cocopeat (T7: solid matrix priming with cocopeat for 48 h) and hormonal priming with GA3 (T5: 100 ppm for 24 h) were found to be significantly superior to other priming treatments as well as untreated seeds. Priming with PEG 6000 (T4: -1.5 MPa for 24 h) was found to be detrimental and registered the least seed indices. The interaction between seed priming treatment and storage conditions was found to significantly influence germination as well as other quality indices of the aged seeds. Results pointed out that germination and most seed indices studied were significantly superior in solid matrix primed seeds involving perlite (T6: solid matrix priming with perlite for 48 h) under both low temperature storage (MTS and Rf.S), as well as ambient storage (Am.S). Solid matrix priming with cocopeat (T7: solid matrix priming with cocopeat for 48 h) and hormonal priming with GA3 (T5: 100 ppm for 24 h), were found to be next best to SMP with perlite under low temperature storage environments viz., MTS and Rf.S. However, unlike in the low temperature storage environments, under ambient storage conditions, bio-priming with P. fluerescens (T8: P. f at 1x106 cfu.ml-1 for 24 h) was found to be next best to SMP with perlite and superior over SMP with cocopeat and hormonal priming with GA3. However, apart from untreated seeds, osmopriming seeds with PEG 6000 (T4: PEG 6000 -1.5 MPa for 24 h), hydration with cold water (T9: Hydration hot water at 50°C for 4 h) as well as with hot water (T10: Hydration cold water soaking for 24 h), were found to be detrimental under low temperature storage (MTS and Rf.S) and ambient storage as well. The results obtained thus point out that loss of germination, vigour and other quality parameters over storage is inevitable. However, the deterioration of seed indices in bitter gourd due to ageing can be slowed down to the maximum extent by storing them under Medium-Term storage (MTS). Storing seeds under refrigerated storage (Rf.S) is highly beneficial compared to storing them under ambient conditions (Am.S). Irrespective of the environment under which the seeds were stored, solid matrix priming of bitter gourd seeds with perlite (48 h) was the best priming treatment as it resulted in significantly superior germination, growth indices of seedlings, biochemical parameters and seed structural changes over untreated seeds. SMP with cocopeat (48 h) and hormonal priming with GA3 (100 ppm for 24 h) were next best to SMP with perlite in bitter gourd. Subjecting seeds of bitter gourd stored either in low temperature environment like medium term storage and refrigerated storage or under ambient environments to solid matrix priming with perlite would result in enhanced germination and seed quality indices. Solid matrix priming with cocopeat or hormonal priming of the aged seeds with GA3 (100 ppm for 24 h) were the next best priming options, if the seeds were stored at low temperature environments. However, in seed stored under ambient environments, bio-priming with P. fluorescens (1x106 cfu.ml-1 for 24 h) would be the best priming option, next to solid matrix priming with perlite.
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    Development of gynoecious genotypes with superior fruit quality in bitter gourd (Momordica Charantia L.) through marker assisted breeding
    (Department of Vegetable Science, College of Agriculture ,Vellanikkara, 2024-02-06) Anju M Sunny.; Pradeepkumar, T
    The doctoral study entitled ‘Development of gynoecious genotypes with superior fruit quality in bitter gourd (Momordica charantia L.) through marker assisted breeding’ was undertaken with the objective of developing gynoecious bitter gourd lines with superior fruit quality through marker assisted breeding and to analyze the genetics of sex expression and fruit quality by generating F2 and back cross population. The work was carried out in the experimental field of the Department of Vegetable Science, College of Agriculture, Vellanikkara. Gynoecious line KAUMCGy- 101, identified from the Department of Vegetable Science, and three monoecious lines viz., Preethi, Priyanka, and MC 133 were used for the hybridization. Six hybrids were produced by crossing gynoecious inbred as both male and female parents. The hybrids were, Preethi × KAU-MCGy-101, Priyanka × KAUMCGy- 101, MC133 × KAU-MCGy-101, KAU-MCGy-101 × Preethi, KAU-MCGy- 101 × Priyanka and KAU-MCGy-101 × MC133. All the hybrids with gynoecious line as male or female parent exhibited high heterosis for earliness attributing traits like days to first female flower opening, node bearing first female flower, and days to first harvest. The traits such as a lower sex ratio and a greater number of fruits per plant contributed to higher yields in these hybrids. The superiority of hybrids having a gynoecious line as a maternal parent is prominent in the present study. The correlation studies revealed a positive correlation between fruit length (0.569), fruit weight (0.612), number of seeds (0.507), and number of harvests (0.619) with yield. While days to first female flower opening (-0.386), node bearing first female flower (-0.244), fruit girth (-0.018), and days to first harvest (-0.285) showed a non-significant negative correlation. Path analysis showed a positive direct effect of fruit weight (0.568), fruit girth (0.155), number of seeds (0.579), days to first harvest (0.009), number of harvests (0.755), and number of fruits per plant (0.036) on yield per plant. The sensory attributes, nutritional potentials, and shelf life of six bitter gourd hybrids and their parents were evaluated. Bitter gourd fruits with white or light green fruits were found to have a more appealing flavor and appearance than dark green fruits, resulting in a higher overall acceptability rating. The hybrid, KAU-MCGy-101 × MC 133 was found superior with 17.05 mg/100g calcium. Preethi and KAUMCGy- 101 × Priyanka exhibited maximum shelf life among the parents and hybrids, respectively. Among the hybrids, the cross KAU-MCGy-101 × Priyanka was superior with respect to yield and quality traits (fruit weight- 172.47 g, number of fruits per plant- 69.67 and yield per plant- 9.15 kg). The genetics of economically significant horticultural traits in the cross KAU-MCGy-101 x Priyanka was studied resorting generation mean analysis. The results of the A, B, C, and D scaling tests indicate that all the traits, except for days to the first female flower opening, do not adhere to an additive dominance model. The significant and positive additive effect (d) was observed for the number of tubercles and number of fruits per plant, indicating that the additive effect of the genes is predominant and that the selection of these traits should be delayed to later generations. Regarding the yield, the interaction effect of dominance x dominance (l) was highly significant, while the other interaction effects were insignificant. Therefore, recurrent selection is necessary for improving this trait. A complementary type of epistasis was observed for the number of fruits per plant, while all other traits were observed to be of the duplicate type. The χ2 analysis for goodness of fit indicated that gynoecy in the line KAUMCGy- 101 is controlled by a single recessive gene (gy-1). The inheritance pattern of fruit color indicates that green fruit color is dominant over white. The present study identified two gynoecious lines from the F2 population namely, KAU-MCGy-101-1 and KAU-MCGy-101-2 (green and light green fruited respectively). These gynoecious lines had better fruit quality and hold immense potential for future breeding programs in bitter gourd improvement. Marker-assisted selection is considered one of the important strategies for identifying different sex forms in crop plants. The present investigation resulted in identifying one SNP primer that could produce polymorphism between the monoecious and gynoecious parents. The six exonic regions with flanking introns of the gene floral homeotic protein AGAMOUS-like (McAG2) and two exonic regions with flanking introns of the gene ACO1 were sequenced by designing exons-specific primer pairs. The primer NETBG2 developed based on the exon 2 region of the gene ACO1 accounts for eight valid SNPs within the sequenced region of the gynoecious parent used in this study. The study confirmed the monogenic recessive nature of gynoecy. The improved gynoecious lines identified will help in developing cultivars with a higher proportion of female flowers. The results of the molecular work revealed the role of the 1-aminocyclopropane-1-carboxylate oxidase (ACO1) gene in the sex expression of the bitter gourd. The SNPs identified can be used for further studies on the SNP-based marker development for identifying gynoecious plants and have the potential in marker assisted breeding based on gynoecy in bitter gourd.
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    Development and evaluation of high yielding, mosaic tolerant backcross progenies in bitter gourd (Momordica charantia L.) variety Preethi using morphological, biochemical and molecular markers
    (Department of Genetics and Plant Breeding, College of Agriculture ,Vellayani, 2024-05-23) Ankitha, M O; KAU; Bindu, M R
    The present research work entitled ‘Development and evaluation of high yielding, mosaic tolerant backcross progenies in bitter gourd (Momordica charantia L.) variety Preethi using morphological, biochemical and molecular markers’ was conducted in the Department of Plant Breeding and Genetics, College of Agriculture, Vellayani and Farming Systems Research Station (FSRS), Sadanandapuram during the year 2020-2023, with an objective to develop high yielding mosaic tolerant backcross progenies in bitter gourd using morphological, biochemical and molecular markers. Thirty three bitter gourd genotypes, including KAU released varieties (2 No’s), NBPGR accessions (13 No’s), and local collections from all over India were used for screening mosaic tolerance. Out of the 33 genotypes, 26 genotypes were Momordica charantia var. charantia and seven were Momordica charantia var. muricata. All these genotypes were artificially inoculated with the three viruses Cucumber Mosaic Virus (CMV), Tomato Leaf Curl New Delhi Virus (ToLCNDV) and Papaya Ringspot virus (PRSV) through wedge grafting. Wedge grafting was done using the infected plant shoots as scion and the collected genotypes as root stock and regrowth from the cotyledonary axis was examined for symptom expression. Out of the 33 genotypes screened, three were highly resistant, four were resistant, five were moderately resistant, six were moderately susceptible, ten were susceptible and five were highly susceptible. The genotypes Lodhi local, Udayagiri local and Therthali local recorded a lowest Vulnerability Index of zero. Molecular markers reported in Cucurbitaceae family were validated for bitter gourd mosaic resistance gene. SSR-11-1 marker for CMV resistance and CAPS marker for Potyvirus resistance gene were used, but no amplification was obtained. Double Antibody Sandwich ELISA (DAS-ELISA) was performed to confirm the resistance reaction of three highly resistant genotypes identified in seedling screening. Optical density (OD) value of the genotypes for the three viruses, CMV, ToLCNDV and PRSV were less than twice the OD value of the un-inoculated healthy plant which confirmed highly resistant disease reaction of genotypes. Molecular confirmation was done by using coat protein primer (Deng primer) specific to the Begomovirus group. Deng primer amplifies coat protein gene of ToLCNDV (520 bp), so that band will be present in only susceptible genotypes and will be absent in resistant ones. Plant defense related enzymes such as peroxidase, polyphenol oxidase and phenyl alanine ammonialyase was estimated and there was increased rate of synthesis of these enzymes in the identified resistant genotypes. So the identified resistant genotypes, Lodhi local, Udayagiri local and Therthali local were used as the donor parent for imparting mosaic resistance into the bitter gourd variety Preethi. Lodhi local is M. charantia var. charantia genotype where as both Udayagiri local and Therthali local are M. charantia var. muricata genotypes. High yielding variety released from KAU viz., ‘Preethi’ was selected as the recurrent parent in the study. Preethi was crossed with the three donor parents and F1s were produced. The F1s were morphologically evaluated with the parents for seventeen characters and it was observed that all the characters of F1 were approximately the average of two parents. All the F1s were backcrossed with Preethi to produce BC1F1 segregants. In the backcross progeny of the cross involving Preethi and Lodhi local, a total of 176 BC1F1 lines were developed. BC1F1 lines were artificially inoculated for their disease reaction. Among the 176 BC1F1 lines, 22 were found to be highly resistant to mosaic disease, 30 were resistant, 30 were moderately resistant, 26 were moderately susceptible, 35 were susceptible and 33 were highly susceptible. Confirmation of resistance was done using DAS- ELISA, Deng primers and estimation of defense enzymes. All the 17 biometrical characters were recorded and the Euclidean distance of the highly resistant BC1F1 lines from the recurrent parent Preethi was calculated using proximity dissimilarity matrix analysis. The 14 BC1F1 lines with high phenotypic similarity to Preethi was backcrossed to develop BC2F1 lines. In the backcross progeny of the cross involving Preethi and Udayagiri local, a total of 170 BC1F1 lines were produced. Among them 15 BC1F1 lines were highly resistant. Resistant reaction of identified 15 BC1F1 was confirmed by DAS-ELISA, molecular screening and biochemical analysis. Euclidean distance of the highly resistant 15 BC1F1 lines from the recurrent parent revealed that eight lines showed similarity with Preethi and they were backcrossed to get BC2F1 lines. A total 147 BC1F1 lines of the cross involving Preethi and Therthali local were screened at seedling stage. Out of the 147 lines, 16 BC1F1 lines were highly resistant. DAS-ELISA, molecular screening using aforementioned Deng primer confirmed the resistant reaction of these lines. Euclidean distance using biometric characters found that, out of 16 highly resistant BC1F1 lines eight lines had close proximity with Preethi. These lines were used to produce BC2F1 lines. The 190 BC2F1 lines of the cross involving Preethi and Lodhi local were screened at seedling stage and in 24 BC2F1 lines, there was absence of virus coat protein band which confirmed the highly resistant disease reaction of the aforementioned lines. The 12 BC2F1 lines with the shortest Euclidean distance and high phenotypic similarity with Preethi were selfed to generate BC2F2 seeds. In the 134 BC2F1 lines of the cross involving Preethi and Udayairi local, seedling screening recorded 17 highly resistant lines. After molecular confirmation of mosaic resistance four BC2F1 lines with close proximity to Preethi were selfed to get BC2F2 seeds. Out of the 143 BC2F1 lines of the cross involving Preethi and Therthali local, 20 BC2F1 lines were highly resistant. The molecular analysis of the 20 BC2F1 lines also confirmed the highly resistant reaction. Four BC2F1 lines with the shortest Euclidean distance was selected and selfed to produce BC2F2 seeds. Although there were BC2F2 seeds of three different crosses, only the BC2F2 seeds of the cross involving Preethi and Lodhi local was carried forward for further backcrossing. This is due to the low yield potential of the backcross progenies of the crosses involving M. charantia var. muricata genotypes. So 206 BC2F2 lines of the cross involving Preethi and Lodhi local were artificially screened at seedling stage for mosaic incidence. Out of the 206 BC2F2 lines, 42 plants were highly resistant to bitter gourd mosaic viruses. The 42 mosaic tolerant backcross inbred lines developed in the study can be carried forward for the development of a mosaic tolerant essentially derived variety (EDV) in the background of high yielding variety Preethi. The backcross progenies obtained in the crosses involving muricata genotypes can be further evaluated for its nutraceutical values.
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    Development and evaluation of high yielding, mosaic tolerant backcross progenies in bitter gourd (Momordica charantia L.) variety Preethi using morphological, biochemical and molecular markers
    (Department of Genetics and Plant Breeding, College of Agriculture, Vellayani, 2024-05-23) Ankitha, M O; KAU; Bindu, M R
    The present research work entitled ‘Development and evaluation of high yielding, mosaic tolerant backcross progenies in bitter gourd (Momordica charantia L.) variety Preethi using morphological, biochemical and molecular markers’ was conducted in the Department of Plant Breeding and Genetics, College of Agriculture, Vellayani and Farming Systems Research Station (FSRS), Sadanandapuram during the year 2020-2023, with an objective to develop high yielding mosaic tolerant backcross progenies in bitter gourd using morphological, biochemical and molecular markers. Thirty three bitter gourd genotypes, including KAU released varieties (2 No’s), NBPGR accessions (13 No’s), and local collections from all over India were used for screening mosaic tolerance. Out of the 33 genotypes, 26 genotypes were Momordica charantia var. charantia and seven were Momordica charantia var. muricata. All these genotypes were artificially inoculated with the three viruses Cucumber Mosaic Virus (CMV), Tomato Leaf Curl New Delhi Virus (ToLCNDV) and Papaya Ringspot virus (PRSV) through wedge grafting. Wedge grafting was done using the infected plant shoots as scion and the collected genotypes as root stock and regrowth from the cotyledonary axis was examined for symptom expression. Out of the 33 genotypes screened, three were highly resistant, four were resistant, five were moderately resistant, six were moderately susceptible, ten were susceptible and five were highly susceptible. The genotypes Lodhi local, Udayagiri local and Therthali local recorded a lowest Vulnerability Index of zero. Molecular markers reported in Cucurbitaceae family were validated for bitter gourd mosaic resistance gene. SSR-11-1 marker for CMV resistance and CAPS marker for Potyvirus resistance gene were used, but no amplification was obtained. Double Antibody Sandwich ELISA (DAS-ELISA) was performed to confirm the resistance reaction of three highly resistant genotypes identified in seedling screening. Optical density (OD) value of the genotypes for the three viruses, CMV, ToLCNDV and PRSV were less than twice the OD value of the un-inoculated healthy plant which confirmed highly resistant disease reaction of genotypes. Molecular confirmation was done by using coat protein primer (Deng primer) specific to the Begomovirus group. Deng primer amplifies coat protein gene of ToLCNDV (520 bp), so that band will be present in only susceptible genotypes and will be absent in resistant ones. Plant defense related enzymes such as peroxidase, polyphenol oxidase and phenyl alanine ammonialyase was estimated and there was increased rate of synthesis of these enzymes in the identified resistant genotypes. So the identified resistant genotypes, Lodhi local, Udayagiri local and Therthali local were used as the donor parent for imparting mosaic resistance into the bitter gourd variety Preethi. Lodhi local is M. charantia var. charantia genotype where as both Udayagiri local and Therthali local are M. charantia var. muricata genotypes. High yielding variety released from KAU viz., ‘Preethi’ was selected as the recurrent parent in the study. Preethi was crossed with the three donor parents and F1s were produced. The F1s were morphologically evaluated with the parents for seventeen characters and it was observed that all the characters of F1 were approximately the average of two parents. All the F1s were backcrossed with Preethi to produce BC1F1 segregants. In the backcross progeny of the cross involving Preethi and Lodhi local, a total of 176 BC1F1 lines were developed. BC1F1 lines were artificially inoculated for their disease reaction. Among the 176 BC1F1 lines, 22 were found to be highly resistant to mosaic disease, 30 were resistant, 30 were moderately resistant, 26 were moderately susceptible, 35 were susceptible and 33 were highly susceptible. Confirmation of resistance was done using DAS- ELISA, Deng primers and estimation of defense enzymes. All the 17 biometrical characters were recorded and the Euclidean distance of the highly resistant BC1F1 lines from the recurrent parent Preethi was calculated using proximity dissimilarity matrix analysis. The 14 BC1F1 lines with high phenotypic similarity to Preethi was backcrossed to develop BC2F1 lines. In the backcross progeny of the cross involving Preethi and Udayagiri local, a total of 170 BC1F1 lines were produced. Among them 15 BC1F1 lines were highly resistant. Resistant reaction of identified 15 BC1F1 was confirmed by DAS-ELISA, molecular screening and biochemical analysis. Euclidean distance of the highly resistant 15 BC1F1 lines from the recurrent parent revealed that eight lines showed similarity with Preethi and they were backcrossed to get BC2F1 lines. A total 147 BC1F1 lines of the cross involving Preethi and Therthali local were screened at seedling stage. Out of the 147 lines, 16 BC1F1 lines were highly resistant. DAS-ELISA, molecular screening using aforementioned Deng primer confirmed the resistant reaction of these lines. Euclidean distance using biometric characters found that, out of 16 highly resistant BC1F1 lines eight lines had close proximity with Preethi. These lines were used to produce BC2F1 lines. The 190 BC2F1 lines of the cross involving Preethi and Lodhi local were screened at seedling stage and in 24 BC2F1 lines, there was absence of virus coat protein band which confirmed the highly resistant disease reaction of the aforementioned lines. The 12 BC2F1 lines with the shortest Euclidean distance and high phenotypic similarity with Preethi were selfed to generate BC2F2 seeds. In the 134 BC2F1 lines of the cross involving Preethi and Udayairi local, seedling screening recorded 17 highly resistant lines. After molecular confirmation of mosaic resistance four BC2F1 lines with close proximity to Preethi were selfed to get BC2F2 seeds. Out of the 143 BC2F1 lines of the cross involving Preethi and Therthali local, 20 BC2F1 lines were highly resistant. The molecular analysis of the 20 BC2F1 lines also confirmed the highly resistant reaction. Four BC2F1 lines with the shortest Euclidean distance was selected and selfed to produce BC2F2 seeds. Although there were BC2F2 seeds of three different crosses, only the BC2F2 seeds of the cross involving Preethi and Lodhi local was carried forward for further backcrossing. This is due to the low yield potential of the backcross progenies of the crosses involving M. charantia var. muricata genotypes. So 206 BC2F2 lines of the cross involving Preethi and Lodhi local were artificially screened at seedling stage for mosaic incidence. Out of the 206 BC2F2 lines, 42 plants were highly resistant to bitter gourd mosaic viruses. The 42 mosaic tolerant backcross inbred lines developed in the study can be carried forward for the development of a mosaic tolerant essentially derived variety (EDV) in the background of high yielding variety Preethi. The backcross progenies obtained in the crosses involving muricata genotypes can be further evaluated for its nutraceutical values.
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    Mapping the QTL for yield traits in bitter gourd (Momordica charantia L.)
    (Centre for Plant Biotechnology and Molecular Biology, College of Agriculture, Vellanikkara, 2022) Lavale Shivaji Ajinath; Deepu Mathew
    Bitter gourd (Momordica charantia), being a rich source of phytonutrients such as carbohydrates, minerals, vitamins, and other medicinal compounds, has a great importance in healthy dietary habits. Breeders always seek to breed bitter gourd varieties for the traits such as early maturity and high yield. However, limited investigations have been made to identify the genetic loci governing yield related traits. Marker assisted selection (MAS) assures the presence of favourable alleles and fast recovery of recurrent parent genome in the cultivar under improvement. The success of MAS mainly depends on the availability of a marker-dense genetic linkage map locating quantitative trait loci (QTL) for the target traits. The present study “Mapping the QTL for yield traits in bitter gourd (Momordica charantia L.)” was carried out during October, 2018 to December, 2021 with the objective to map the quantitative trait loci and to develop chromosome-wise maps for the yield traits in bitter gourd. To develop the mapping population, high yielding bitter gourd cultivar Priyanka (Momordica charantia var. charantia) and a wild bitter gourd accession IC634896 (M. charantia var. muricata), were used as parents. A set of 450 microsatellites were screened for polymorphism using genomic DNA of parents and 47 were found polymorphic. Bitter gourd genome (GenBank acc. no. GCA_013281855.1) was scanned and new hypervariable microsatellites were identified using Genome wide Microsatellite Analysing Tool (GMATo) and named as KAUBG_n where n is a serial number. From the 75 microsatellites identified, 69 were validated through successful PCR amplification and 38 among them were polymorphic between the parents. This led to the development of a set of 85 markers polymorphic between the parents. Crosses were made between the parental lines and hybrids from the cross Priyanka × IC634896 yielded more number of fruits and total fruit produce compared to the reciprocal hybrid. An F2:3 population was developed through single seed descent method from the cross Priyanka × IC634896. A panel of 200 F2:3 plants were evaluated for twenty seven traits, including fruit-, flower-, seed-, vine-, and leaf-related traits, contributing directly or indirectly to the total yield. Wide variation was observed among the F2:3 plants for the traits studied. A group of ninety plants was selected from 200 F2:3 plants such that they represent the variation of the base population. Genomic DNA of these plants were genotyped using 85 polymorphic markers. Genotypic data from the screening of 85 markers in the mapping population were used to generate a linkage map spanning 1287.99 cM distance across eleven linkage groups (LGs) corresponding to eleven chromosomes, using IciMapping software. LG 7 (28 markers) consisted of maximum number of markers followed by LG 2 and LG 9, each having 11 markers. LG 1 had 10 markers whereas LG 3, 4 and 8 had seven markers each. LG 5, 6, 10 and 11 had only one marker each. LG 7 covered maximum map distance of 384.19 cM where LG 8 covered least map distance of 68.58 cM. The genetic map and phenotypic data were used to generate the QTL maps, using Inclusive Composite Interval Mapping (ICIM) method to locate twenty seven traits on Momordica genome. Sixty QTL, including 37 major QTL with LOD values ranging from 3.1 to 15.2, explaining 1.8 to 35.9 per cent of the phenotypic variation were identified for 24 traits, on seven chromosomes. Twenty three QTL were identified for fruit-traits with LOD values ranging from 3.1 to 7.6, explaining 5.5 to 35.9 per cent of phenotypic variation. Thirteen QTL were identified for flower-related traits with LOD value ranging from 3.1 to 15.2, explaining 7.0 to 26.0 per cent of phenotypic variation. Seven QTL each were identified for seed and leaf-related traits with LOD values ranging from 3.2 to 10.8 and 3.5 to 6.5, explaining 5.6 to 26.3 and 3.2 to 15.8 per cent of phenotypic variation, respectively. Ten QTL were identified for vine-related traits with 3.2 to 8.7 LOD values and explaining 1.8 to 17.6 per cent of phenotypic variation. Single marker analysis was performed to identify markers co-segregating with the yield contributing traits. There were 129 hits for the marker-trait association with LOD values more than 3.0, explaining 11.62 to 29.34 per cent of the phenotypic variation. Using the least and best performing F2:3 plants, markers S13, KAUBG_5 and KAUBG_11 were validated for co-segregation with fruit breadth, first pistillate flower node, and number of pistillate flowers and fruits per plant, respectively. This study gives insights into the relative locations of microsatellites and major effect QTL for yield traits in Momordica genome. QTL with shorter marker interval (qFrtL-8-1, qDPF-3-1, qDSF-3-1, qDSF-7-1, qFrtShp-8-1) can be directly used in MAS for improving yield characters. Linkage observed between microsatellites identified in this study with yield traits signifies their importance in further fine mapping as well as marker assisted selection. The linkage map constructed in this study, being the first with microsatellites from Momordica genome, paves the path for comparative and consensus map generation with other marker types. Further, fine mapping using markers within the identified QTL hotspots can lead to possible identification and cloning of genes underlying the yield traits.
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    Molecular characterization of gynoecy in bitter gourd (Momordica charantia L.)
    (Department of Plant Biotechnology, College of Agriculture, Vellanikkara, 2021) Nivethitha, B; Pradeepkumar, T
    Bitter gourd (Momordica charantia L.) is an important vegetable of tropics and sub-tropics of Asia belonging to Cucurbitaceae family. The immature fruits of bitter gourd are valued for its culinary and medicinal importance. Having highest amount of ascorbic acid and iron content, it is considered to be the most nutritive among all the cucurbitaceous vegetables. Heterosis is well exploited in bitter gourd for early maturity, increased yield and other agronomic traits through development of hybrids. However, the production of hybrids is labour-intensive involving manual bagging and hand pollination, thereby increasing the cost of seed production. The predominant sex form of bitter gourd is monoecious which bears separate male and female flowers on the same plant. However, gynoecious type bearing only female flowers were also reported in few locations of India, Japan and China. These gynoecious bitter gourd lines can be exploited as female parent to make hybrid seed production economical and easier, as it eliminates the need for emasculation and assisted pollination. It also aids in maintaining the genetic purity of hybrids and helps in harnessing the benefit of hybrid vigour including early maturity and high yield. Usually, the sex expression in cucurbits is highly influenced by environment and hormones, which makes the early phenotypic identification of gynoecy challenging. Identification of molecular markers tightly linked to gynoecy trait would ease the identification of gynoecious line in breeding programmes. In this study, 20 putative candidate genes governing sex expression were selected from different literatures and the gene sequences were retrieved from bitter gourd and cucumber genome assembly in NCBI GenBank database. Four ethylene biosynthesis genes (ACO1, ACS2, ACS3. ACS4 and ACS7), three MADS-box transcription factor encoding genes (AG6, MADS-boxTF23 and McAG2), one auxin related gene (CsARF5) and 12 WRKY transcription factor encoding genes comprise the list. A total of 20 gene-specific primer sets were designed from the selected region of each of the 20 genes. Genomic DNA was isolated from monoecious genotype, MC-136 and gynoecious, KAUMCGy-101. Efforts were made to amplify all the 20genes, however, only seven gene-specific primer sets designed from bitter gourd produced PCR amplification. The amplicons of expected product size from both the samples were sequenced. Sequence variation analysis was done by comparing the monoecious and gynoecious sequences generated to the reference sequence (monoecious) available in NCBI database for bitter gourd. Six valid variations including three SNPs and three In/Dels were found in AG6 and McAG2. All the variations except SNP1 of AG6 were present in non-coding regions. In/Del of 48 bp ([TC] 24 ) in AG6 gene caused a significant difference in the number of ‘TC’ repeats in the sequences which was used for the development of SSR marker. The marker showed clear polymorphism between monoecious and gynoecious genotypes used. It was further validated in three other monoecious lines namely, Priya, Priyanka and the wild type M. charantia var. muricata. Expected banding pattern showing polymorphism for gynoecious line was obtained for all the three monoecious lines screened. Thus, the study identified two potential candidate genes, AG6 and McAG2 for sex expression in bitter gourd. The SSR marker developed needs to be validated in large number of population and more number of varieties to confirm its use as a reliable polymorphic marker in marker-assisted selection of gynoecious lines of bitter gourd.
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    Homeostatic analysis of components of genetic variance and inheritance of fruit colour, fruit shape and bitterness in bitter gourd (Momordica charantia L.)
    (Department of Olericulture, College of Horticulture, Vellanikkara, 1989) Abdul Vahab, M; Gopalakrishnan, P K